In this study, similar results were found in blood pressure, bleeding time and plasma cGMP levels of the injury phase and the matrix expansion phase in acute anti-thy1 glomerulonephritis. In the interest of simplicity, they are presented together.

As shown in Figure 5, plasma cGMP levels were significantly higher in the untreated aGN group than those in the normal controls (4130±650 fmol/ml vs. 2120±260 fmol/ml), and were elevated significantly further by Bay 41-2272 treatment (6610±810 fmol/ml).

Summing up, Bay 41-2272 treatment caused a decrease in systolic blood pressure, a prolongation of bleeding time and an increase in the plasma cGMP levels, which proves the in vivo bioavailability of the drug through stimulating cGMP synthesis.

The aGN+Bay 41-2272 group was treated for 6 days before and 1 day after the induction of acute anti-thy1 glomerulonephritis. The experiment was ended 1 day after disease induction. There was no difference in the body weight at the end of the experiment among the three groups (Control 253±3 g, aGN 250±5 g and aGN+Bay 41-2272 255±7 g).

Proteinuria

The proteinuria of diseased rats was significantly higher than that in the control group (14±2 mg/day, p<0.01 vs. aGN and aGN+Bay 41-2272). Treatment with Bay 41-2272 before anti-thy1 antibody-induced mesangial cell lysis reduced proteinuria slightly, but not significantly, when compared to the aGN group (31±4 mg/day vs. 53±10 mg/day, p>0.05).

Mesangial cell lysis

As shown in Figure 6, anti-thy1 antibody injection resulted in a rapid drop in glomerular cell number. Cell nuclei counts averaged 56.3±1.1 per glomerular section in the control group, whereas the aGN group’s and the aGN+Bay 41-2272 group’s cell counts were significantly reduced to 44±1.1 and 44.3±1.1 per glomerular section, respectively. There was no difference between the aGN group and the aGN+Bay 41-2272 group. This result indicates that Bay 41-2272 has no effect on mesangial cell injury 1 day after anti-thy1 glomerulonephritis induction.

As shown in Figure 7, glomerular iNOS mRNA expression increased significantly in the aGN group (263±38%) as compared to the control group (100±28%). This increase was not significantly changed in the Bay 41-2272 treated group (226±26%).

Figure 7: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular iNOS mRNA expression 1 day after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the normal control group. (**p<0.01 vs. Control)

As shown in Figure 8, glomerular NO production was in line with the iNOS mRNA expression. Compared to the control group (2.1±0.3 nmol/ml), the basal NO synthesis of isolated glomeruli was significantly elevated in the aGN group (6.9±0.5 nmol/ml) and the aGN+Bay 41-2272 group (6.9±1.7 nmol/ml) 1 day after disease induction. LPS stimulated the glomerular NO production significantly in the aGN group (49.1±4.7 nmol/ml) and the aGN+Bay 41.2272 group (50.6±8.8 nmol/ml), in contrast to the control group (9.3±0.9 nmol/ml). And there was no difference between the aGN and the Bay 41-2272-treated aGN group.

Figure 8: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular basal and LPS-stimulated NO production 1 day after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. Nitrite as indicator of NO production was determined by the Griess reaction. Glomeruli were harvested from individual animals and cultured at a density of 2000 per ml for 48 hours. (***p<0.001 and **p<0.01 vs. Control)

Glomerular eNOS-NO-cGMP signaling cascade

As shown in Figure 9, there was no difference in eNOS mRNA expression among the three groups (control 100±15%, aGN 145±23%, aGN+Bay 41-2272 100±14%, P>0.05). The induction of anti-thy1 glomerulonephritis did not significantly change eNOS expression.

Figure 9: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular eNOS mRNA expression 1 day after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the normal control group.

Figure 10: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular alpha1 sGC and beta1 sGC mRNA expression 1 day after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the normal control group.(***p<0.001 vs. Control)

Figure 11: Effects of Bay 41-2272 (+Bay 41-2272) on basal and DEA/NO stimulated glomerular cGMP production 1 day after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. cGMP generation was measured by ELISA in glomeruli harvested from individual animals in the presence or absence of the NO donor DEA/NO. (**p<0.01 vs. Control)

Taken together, the results from protocol 1 consistently demonstrate that the expression and activity of the NO-cGMP pathway are dramatically impaired one day after anti-thy1 glomerulonephritis induction. Treatment with Bay 41-2272 could not stimulate glomerular cGMP synthesis and hence was unable to affect the mesangial cell lysis in the injury phase of acute anti-thy1 glomerulonephritis.

Bay 41-2272 treatment was begun 1 day after and continued 7 days after the induction of acute anti-thy1 glomerulonephritis. The experiment ended 7 days after disease induction. There was no difference in the body weight at the end of the experiment among the three groups (Control 231±14 g, aGN 240±11 g and aGN+Bay 41-2272 240±12 g).

Figure 13: Effects of Bay 41-2272 (+Bay 41-2272) on markers of glomerular matrix expansion, including matrix score, TGF-beta1, fibronectin and PAI-1 production, 7 days after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. Matrix expansion was scored on PAS-stained slides. Glomeruli were harvested from individual animals and cultured at a density of 2000 per ml for 48 hours. (***p<0.001, **p<0.01, *p<0.05 vs. aGN)

Glomerular eNOS-NO-cGMP signaling cascade

As shown in Figure 14, compared to the aGN group (72±10%), Bay 41-2272 treatment significantly up-regulated eNOS mRNA expression (125±20%). There was no significant difference in eNOS mRNA between the control group (100±10%) and the untreated aGN group.

Figure 14: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular eNOS mRNA expression 7 days after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the normal control group. (*p<0.05 vs. aGN)

As shown in Figure 15, compared to the normal controls (alpha1 sGC 100±14% and beta1 sGC 100±15%), sGC mRNA expression in the untreated aGN group (alpha1 sGC 563±76% and beta1 sGC 330±31%) increased significantly after disease induction. Treatment with Bay 41-2272 further up-regulated sGC mRNA expression (alpha1 sGC 1758±544% and beta1 sGC 1218±463%) to a greater extent than in the aGN group. As is consistent with the sGC mRNA expression, basal and NO-stimulated glomerular cGMP production was significantly elevated in nephritic animals (382±156 fmol/well and 38011±17528 fmol/well) as compared to normal controls (basal cGMP production: 16±3 fmol/well, NO-stimulated cGMP production: 195±33 fmol/well). Although basal glomerular cGMP production was similar in treated (381±98 fmol/well) and untreated nephritic animals, glomerular NO-stimulated cGMP production was significantly further increased in the Bay 41-2272-treated animals (181969±114910 fmol/well).

Figure 15: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular sGC mRNA expression and sGC activity to produce cGMP 7 days after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the normal control group. cGMP generation was measured by ELISA in glomeruli harvested from individual animals in the presence or absence of the NO donor DEA/NO. (***p<0.001 and *p<0.05 vs. Control, #p<0.05 vs. aGN)

Mechanisms of Bay 41-2272’s renoprotective effects
Blood pressure

As shown in Figure 3, Bay 41-2272 reduced systolic blood pressure significantly, as compared to control and untreated nephritic animals.

TGF-beta1 production in vitro

As shown in Figure 16, 48 hours of exposure of glomeruli to Bay 41-2272 induced a dose-dependent decrease in glomerular TGF-beta1 production. In normal glomeruli, 0.1 µM, 1 µM, 5 µM and 10 µM Bay 41-2272 reduced TGF-beta1 production by 16%, 24%, 28% and 26%, and in day 7 anti-thy1 nephritic glomeruli, TGF-beta1 production were reduced by 10%, 15%, 25% and 21%, respectively.

Figure 16: In vitro effects of Bay 41-2272 on TGF-beta1 production in glomeruli isolated from control and nephritic rats 7 days after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. Glomeruli were cultured at a density of 2000 per ml for 48 hours in the presence of Bay 41-2272 in a concentration sequence of 0.1 µM, 1 µM, 5 µM and 10 µM. (*p<0.05 vs. glomeruli of Control and aGN without Bay 41-2272)

Glomerular platelet deposition

As shown in Figure 17, fibrinogen staining was hardly found in the control group. In contrast to the untreated aGN group (45±6% staining-positive area per glomerulus), Bay 41-2272 treatment (29±3% staining-positive area per glomerulus) significantly reduced fibrinogen deposition, which indicates platelet aggregation in the glomeruli. Combined with the prolonged bleeding time, this indicates that Bay 41-2272 has an anti-platelet effect through elevated cGMP synthesis.

Figure 17: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular fibrinogen deposition 7 days after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. Glomerular fibrinogen deposition is expressed as the percentage of fibrinogen-positive area per glomerular cross-section. (*p<0.05 vs. aGN)

Glomerular macrophage infiltration

As shown in Figure 18, the nephritic groups had more glomerular macrophage infiltration than the control group (0.8±0.2 cells per glomerular section). Glomerular macrophage infiltration was significantly inhibited by Bay 41-2272 treatment (6.2±0.5 cells per glomerular section), in contrast to the aGN group (9.2±1.2 cells per glomerular section). Although there was no significant difference in P-selectin mRNA expression among the three groups, Bay 41-2272 treatment (85±12%) slightly inhibited glomerular P-selectin mRNA expression, as compared to the aGN group (132±25%).

Figure 18: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular ED1-positive cell infiltration and P-selectin mRNA expression 7 days after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. Glomerular macrophage infiltration is expressed as number of ED1-positive cells per glomerular cross-section. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the normal control group. (*p<0.05 vs. aGN)

Taken together, the results of protocol 2 show that the NO-cGMP pathway is significantly up-regulated in the matrix expansion phase of acute anti-thy1 glomerulonephritis. Bay 41-2272 treatment further increased sGC expression and activity to produce cGMP, which limited the fibrotic parameters characterized in the matrix expansion phase of acute anti-thy1 glomerulonephritis.

Treatments with Bay 41-2272 and hydralazine were started 7 days after the induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). The experiment ended 16 weeks after disease induction. There was no difference in body weights at the beginning and the end of the experiment among the three cGS groups. At the end of the experiment, the final body weights in all diseased groups were significantly lower than those in the control groups.

Table 7: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on rats’ body weights at the beginning and the end of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. (*p<0.05 vs. Control)

Groups

2-K Control

1-K Control

cGS

cGS+Bay 41-2272

cGS+Hydralazine

Begin

256±3 g

230±8 g

220±2 g

224±2 g

219±3 g

End

575±21 g

570±24 g

488±15 g*

482±11 g*

464±13 g*

Systolic blood pressure

As shown in Figure 19, the model of anti-thy1-induced chronic glomerulosclerosis was characterized by a significant increase in systolic blood pressure 8 weeks and 16 weeks after disease induction (134±2 mmHg and 135±1 mmHg, respectively), compared to 2-K Control (129±2 mmHg and 122±4 mmHg) and 1-K Control (122±4 mmHg and 122±5 mmHg). Bay 41-2272 treatment lowered blood pressure significantly to 116±3 mmHg in week 8 and 116±2 mmHg in week 16 after anti-thy1 antibody-injection, in comparison to the untreated cGS groups. Hydralazine administration reduced also systolic blood pressure significantly to 109±4 mmHg and 110±3 mmHg, respectively. Due to the absence of a significant difference between the Bay 41-2272-treated group and the hydralazine-treated group, hydralazine was a good control drug to observe the blood pressure-independent effect of Bay 41-2272.

As shown in Figure 20, all diseased rats were randomized at the beginning of the protocol, so that every diseased group had similar proteinuria (cGS 148±8 mg/day, cGS+Bay 41-2272 146±7 mg/day, cGS+Hydralazine 148±10 mg/day), and proteinuria was significantly elevated in all diseased groups, in contrast to the 2-K and 1-K Controls (20±4 mg/day and 17±2 mg/day). During the experiment, proteinuria gradually increased in the three diseased groups, indicating a progressive chronic glomerulosclerosis model. At the end of the protocol, proteinuria was reduced slightly by Bay 41-2272 treatment (cGS+Bay 41-2272 422±42 mg/day, p>0.05 vs. cGS 476±56 mg/day and cGS+Hydralazine 451±70 mg/day), although not significantly.

Figure 20: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on time course of proteinuria after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. Urine was collected for 24 hours using metabolic cages.

Markers of tubulointerstitial matrix accumulation
Matrix score

As shown in Figure 21, compared to the 2-K (0.04±0.004) and the 1-K Control (0.1±0.02), injection of anti-thy1 antibody induced significant tubulointerstitial matrix protein deposition in the cGS group (2.49±0.22). Bay 41-2272 treatment reduced tubulointerstitial matrix protein accumulation significantly (-35.1%), in contrast to hydralazine treatment, which lowered matrix protein accumulation only slightly but not significantly (-9.2%), suggesting that Bay 41-2272 had a blood pressure-independent effect on tubulointerstitial matrix protein accumulation.

Figure 22: Effects of Bay 41-2272 and hydralazine on the histological picture 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatment was started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Shown are characteristic PAS-stained renal sections from A) a non-diseased animal without (2-K Control) and B) with uni-nephrectomy (1-K Control), and C) an animal with anti-thy1-induced chronic glomerulosclerosis without treatment and with Bay 41-2272 (D) or with hydralazine treatment (E) (Magnification x200).

Protein and mRNA expression of TGF-beta1, fibronectin and PAI-1

Figure 23: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on tubulointerstitial TGF-beta1, fibronectin and PAI-1 protein and mRNA expression 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. Matrix protein production was determined in extensively minced individual cortical tissues cultured at a density of 10 mg/ml for 48 hours. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the untreated chronic anti-thy1 animals. (***p<0.001, **p<0.01 and *p<0.05 vs. cGS, ###p<0.001 and #p<0.05 vs. cGS+Hydralazine)

Figure 25: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on markers of renal function 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatment was started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. (**p<0.01 and *p<0.05 vs. cGS, #p<0.05 vs cGS+Hydralazine)

Tubulointerstitial eNOS-NO-cGMP signaling cascade

As shown in Figure 26, although there was no significant difference among the three diseased groups (cGS: 100±7%, cGS+Bay 41-2272: 132±14% and cGS+Hydralazine: 100±15%), in comparison to the 1-K and 2-K Control groups (70±12% and 73±9%), eNOS mRNA expression was elevated in diseased animals, especially in the cGS+Bay 41-2272 group.

Figure 26: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on tubulointerstitial eNOS mRNA expression 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the untreated chronic anti-thy1 animals. (*p<0.05 vs. Control)

As shown in Figure 27, in comparison to the 2-K (32±16% and 34±7%) and 1-K (39±16% and 29±7%) Control groups, anti-thy1-induced chronic glomerulosclerosis showed a marked 2.8-fold and 3.2-fold increase in the mRNA expression of the tubulointerstitial alpha1- and beta1 sGC. While basal tubulointerstitial cGMP production did not differ between the untreated diseased group (19±1.48 fmol/well) and the control groups (2-K 24±2 fmol/well and 1-K 22.5±4.5 fmol/well), the NO-stimulated cGMP production was 2.7-fold higher in the chronic anti-thy1 group than in 2-K Control (115±16 fmol/well) and 1-K Control group (92±6 fmol/well). Compared to the untreated diseased animals, administration of Bay 41-2272 did not significantly alter the expression of the sGC (alpha1 80±16% and beta1 95±17%), but resulted in a significantly higher NO-stimulated tubulointerstitial cGMP production (534±126 fmol/well vs. 282±41 fmol/well), while treatment with hydralazine did not significantly affect mRNA expression and activity of sGC (alpha1 116±15%, beta1 118±17% and NO-stimulated cGMP levels 206±37 fmol/well).

Figure 27: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on tubulointerstitial sGC mRNA expression and activity to produce cGMP 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the untreated chronic anti-thy1 animals. cGMP generation was measured by ELISA in cortical tissue from individual animals in the presence or absence of the NO donor DEA/NO. (**p<0.01 vs. cGS+Hydralazine, *p<0.05 vs. Control, #p<0.05 vs. cGS)

Figure 28: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on glomerular sGC activity to produce cGMP 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. cGMP generation was measured by ELISA in glomeruli harvested from individual animals in the presence or absence of the NO donor DEA/NO. (**p<0.01 and ##p<0.01 vs. cGS and cGS+Hydralazine)

Renal macrophage infiltration

As shown in Figure 29, the diseased groups had more cortical and glomerular macrophage infiltration than the 2-K (9.17±0.4 and 0.7±0.14 ED1-positive cells/section) and 1-K (7.86±1.25 and 0.7±0.21 ED1-positive cells/section) Control groups. Cortical and glomerular macrophage infiltration were significantly inhibited by Bay 41-2272 treatment (-39.7% and -46.7%), in contrast to cGS (58±5 and 2.55±0.33 ED1-positive cells/section), while hydralazine treatment did not affect macrophage infiltration (51±5 and 2.58±0.32 ED1-positive cells/section).

Figure 29: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on cortical and glomerular ED1-positive cells infiltration 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. Data are expressed as ED1-positive cells per cortical section observed at x200 magnification and per glomerular cross-section. (**p<0.01 vs. cGS, ##p<0.01 and #p<0.05 vs. cGS+Hydralazine)

As shown in Figure 30, and as it is consistent with macrophage infiltration, the untreated cGS group (100±15%) had significantly more tubulointerstitial P-selectin mRNA expression, which is important for macrophage infiltration, than the 2-K (8±1%) and 1-K (13±2%) Control groups. Bay 41-2272 administration inhibited tubulointerstitial P-selectin mRNA expression significantly (-47%) as compared to cGS, while hydralazine only slightly lowered P-selectin mRNA expression (-19%).

Figure 30: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on tubulointerstitial P-selectin mRNA expression 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the untreated chronic anti-thy1 animals. (*p<0.05 vs. cGS)